Isoelectric Point in Food ProteinsAug. 23, 2017[include the title and date][Sections prepared before lab]Introduction:The purpose of this experiment, is determine the isoelectric point (pI) in proteins.Proteins are made of amino acids that possess various functional groups. These functional groupsdo include both weak acids and bases. The acids and bases are used to determine the isoelectricpoint of a molecule, that carries no net electrical charge and is at a neutral state. The purpose ofdetermining a protein’s pI, is for the usage of the characteristics that it can provide. Determiningthe pI of a product can begin the process of being able to analyze the methods of improving aprotein’s identification in different work flows (Audain el la. 2015). There can be consequencesresulting from a product that is based on when a product does reach around its pI range. Forexample, wheat proteins give the most strength and elasticity when it has reached or is near it’sisoelectric point (Hazen 2006). The properties of the the wheat protein can provide, can becommonly used in many ways in a bakery setting. As the isoelectric point of a product is near,the elasticity will increase, staying at a neutral pH level. The experiment conducted during lab,determined the pI in whey protein isolate. In whey proteins, the outcome will determine therange of when the protein is most soluble.Materials and Methods:During the experiment, my group worked with the protein whey protein isolate. At thestart of the procedure, we used two 140 mL beakers filled with a 50 mL protein solutioncontaining 10 mg/mL whey protein isolate mixed into Deionized water. We used two separateprotein solutions, one specifically for acid and the other for bases. Before we began changing thepH levels of the proteins, we first tested the original pH level with the pH meter (DenverInstrument). After determining the protein’s pH level, we were ready to begin the process ofobtaining all 12 different desired pH levels.The first beaker that was specifically for adjusting the protein solutions to acidic pH’s to6, 5, 4.5, 4, 3, and 2 with HCl. As for the second beaker that was specifically for adjusting theprotein solutions to alkaline pH’s to 7, 8, 9, 10, 11, and 12 with NaOH. It was helpful to use astirring plate to constantly stir the solution to easily reach the desired pH level. Once eachtargeted pH level was reached, 1 mL of the solution was transferred over to two clean test tubes.This now gives us two trials and it represents the total protein content. Also, 1.5 mL of the sameprotein solution was transferred to two 2.0 mL centrifuge tubes. The protein solutions in thecentrifuge tubes were centrifuged (Genesys 2) at 2,000 rpm for 10 minutes total. Once theproduct has been completely separated, 1 mL of the supernatant was transferred over to a cleantest tube. This now also represents the total protein content. The protein concentration ismeasured by using the Biuret method by combining the protein solutions in each test tube alongwith 4 mL of Biuret reagent. Once the reagent has been added, the solution sat at roomtemperature for 30 minutes before reading the absorbance. The absorbance was read at 540 nm.[Sections completed during the lab]Results and Discussion:1Table 1. The concentration and solubility of protein found in the respective pH values.[Make sure to include tables and figures captions. Table captions should be above the tables.Figures captions should be below the figures. Table and figure captions should be able to standalone, meaning that it should include all the information needed to understand the table or figure]pH Actual pHA540 beforecentrifugationA540 aftercentrifugationSolubility (%)[include the unit]Trail 1 Trail 12 2.00 0.437 0.454 1043 3.00 0.433 0.437 1004 3.99 0.442 0.367 834.5 4.50 0.396 0.327 82.505 5.00 0.406 0.334 82.306 6.03 0.448 0.449 1007 7.01 0.402 0.413 1008 7.99 0.480 0.357 74.309 8.97 0.407 0.359 8810 10.01 0.380 0.371 97.6011 11.01 0.371 0.347 93.5012 12.00 0.380 0.326 88.40[Sections completed during or after the lab]Graph 1. Solubility of whey protein isolate at various pH[Make sure to include tables and figures captions.]Based on these results, the lowest point is supposed to indicate that it is the wheyprotein’s pI. The pI indicates at a certain pH that the protein’s minimum solubility value. In theresults obtained from, there happened to be 3 possible pI points based on Graph 1. The pI point isindicated at the pH 4, 5, and 8. The possible source of error that could be indicated would be thepH probe that was used in the experiment. When determining the various levels of pH, the couldhave been an error when using the probe. Secondly, another possible error could be the60%70%80%90%100%110%0 2 4 6 8 10 12 14Solubility (%)pH[Title and unit][Title and unit,The unit of pH and absorbanceis 1. So no need to specify]2possibility of going back and forward from acid to bases. Recall from memory, there might havebeen an incident where the pH was trying to be lowered, it dropped significantly to a differentacidic pH, causing us to reverse back to determine the desired pH value. Thirdly, anotherpossible source of error can be due to the mishandling of products when conducting theexperiment.Discussion:From the information that was collected shown in table 1, the value that indicated thatthere was a decrease in solubility was during the pH range of 4-5 and 8. This does make a largedifferent from these two ranges as one is clearly in an acidic area and the other is in the basearea. Based on an experiment conducted by Pelegrine and Gasparetto, they tested the wheyprotein solubility with temperature and pH. The temperature ranged from 40°C – 60°C with a pHrange of 3.50 – 7.80. They were able to determine that the pI was found at 4.5 pH. Any pH valuesbelow and above that range, the solubility increased due to the conditions of the proteins havingpositive or negative net charges, and the proteins also has interacted with water. Specifically, atthe pI of 4.5 pH, the solubility decreased with the temperature due to the bonds reducing theprotein-water interactions. It was also observed that at the neutrality pH of 6.8, the solubility hadalso decrease with the temperature, showing that the thermal protein denaturation had occurred.Similarly, the results showed quite similarities with the results found in our experiment. Theother factor that isn’t involved in our experiment, would be the heat factor. The possible factorsinvolved could still be the possible sources of error, as mentioned earlier.It is possible for another property of proteins to be altered into a gel formation. Based onan experiment conducted by Ju and Kailara in 1998, whey protein isolate was tested with heat,protease, calcium salt, and acidulant at different pH ranges to determine the gel formation. Gelsbegan to form at the pH of 4.6 and it was found to be significantly stronger than other gels thatare formed at a pI of 5.2. It was found that the protein gelation process can be completed in twovarious stages. The first would include the pH causing aggregation in the protein molecules.Secondly, the heat, enzyme, salt, and acidulant independently different forms of aggregates. Theheat and salt also influences the proteins to cause the result of gelling. In a different experimentby Kirkwood el al. in 2015, the pI found played a role in determining the protein crystallization.The major factor that makes a difference in the case studies is the pH factor. When obtaining thepH levels, it has to be done in a specific way, otherwise the collected data could be skewed. Thedifferent variables used in each experiment, shows that there are many ways to manipulate theformation of the protein aggregates and other properties of gels.References:[follow the format of Journal of Food Science: http://www.ift.org/knowledge-center/read-iftpublications/journal-of-food-science/authors-corner/jfs-authorguidelines.aspx#FormattingReferences]Audain, E., Ramos, Y,. Hermjakob, H., Flower, D. R., Perez-Riverol, Y. (2015). AccurateEstimation of Isoelectric Point of Protein and Peptide Based on Amino Acid Sequence.Bioinformatics Advance Access, 21: 1-7.Hazen, C. (2006). The Power of Protein. Natural Products Insider. Retrieved fromhttp://www.naturalproductsinsider.com/articles/2006/06/the-power-of-proteinnbsp.aspx3Ju, Z. Y., Kilara, A. (1998). Gelation of pH-Aggregated Whey Protein Isolate Solution Inducedby Heat, Protease, Calcium Salt, and Acidulant. J. Agric. Food Chem, 46, 1890-1835.Kirkwood, J., Hargreaves, D., O’Keefe S., Wilson J. (2015). Using Isoelectric Point toDetermine the pH for Initial Protein Crystallization Trails. Bioinformatics, 21, 1-8.Pelegrine, D. H. G., Gasparetto, C. A. (2004) Whey Proteins solubility as Function ofTemperature and pH. Lebensm. Wiss. u.-Technol, 38, 77-80.
Lab 5. Food Pigments: Anthocyanins
Learning objective:
Practice extraction of anthocyanins from food materials
Utilize UV/Vis spectrophotometer to quantify pigment content in various foods
Observe and understand the effect of pH on anthocyanin color
5.1 Introduction:
Anthocyanins are among the most important groups of plant pigments. They are present in almost all the higher plants and are found in all parts of the plant. They are a highly desirable substitute for synthetic food colorants although their instability (to temperature, light, etc.) imposes some limitation on their use in food products.
Anthocyanins are glycosylated, polyhydroxy and polymethoxy derivatives of 2-phenylbenzo pyrylium (flavylium) salts. In many cases, the sugar residues are acylated by p-coumaric, caffeic, ferulic, or sirapic acids and sometimes by p-hydroxybenzoic, malonic, or acetic acids. The acyl substituents are usually bonded to the C-3 sugar. The most common sugars are monosaccharides, i.e., glucose, galactose, rhamnose and arabinose. The most frequently encountered anthocyanidins are pelargonidin, cyaniding, peonidin, delphinidin, malvidin, and petunidin.
Pelargonidin Cyanidin
Peonidin Delphinidin
Malvidin Petunidin
The first step in the separation of individual anthocyanins is the preparation of a crude pigment extract. The separation of pigments can be achieved by thin layer or other types of chromatography.
5.2 Materials and Methods
5.2.1 Materials. Food samples, 10% Citric acid in ethanol (~pH 2.0) (Check pH), Cheese cloth, Whatman #4 filter paper, Potassium chloride buffer (0.025 M, pH 1.0), Sodium acetate buffer (0.4 M, pH 4.5), Spectrophotometer.
5.2.2 Methods:
Prepare the Solutions
Solutions
M.W. (g/mol)
Per 50 mL
Final pH
10% Citric acid in 70% ethanol (~pH 2.0)
5 g citric acid in 45 mL 70% ethanol
Potassium chloride buffer (0.025 M, pH 1.0)
74.56
0.0932 g KCl
Sodium acetate buffer (0.4 M, pH 4.5)
82.03
1.6406 g CH₃COONa
Anthocyanin Extraction
Weigh 5 g (record exact weight) of the fruit sample into a mortar.
Add 10 mL of 10% citric acid monohydrate in 70 % ethanol.
Thoroughly crush the fruit with a pestle. Extract for 20 minutes and stir every 5 minutes.
Filter through cheese cloth onto a Whatman #4 filter paper and into a 25 mL graduated cylinder.
Record the total extract volume and the color of the filtrate in each sample.
Sample 1
Sample 2
Sample 3
Sample wt (g)
Vol. of extract (mL)
Color of extract
Estimation of Total Anthocyanins Content (AOAC Official Method 2005.02):
Prepare a 1:10 dilution (0.5 mL + 4.5 mL) of the extract with buffer solutions of pH 1.0 and 4.5, respectively. Measure the absorbance at 530 nm using the spectrophotometer. Estimate the amount of total anthocyanins by the equation:
mg anthocyanins per mL extract = [(Abs530nm @ pH 1.0) – (Abs530 nm @ pH 4.5)]× 0.0096* × dilution factor
*Based on theabsorptivity value (32,000 M-1cm-1) for Pelargondin (M.W 306.7) @ 530 nm.
Knowing the total volume of extract and the weight of the sample used for extraction, estimate the anthocyanin content and express it as mg pelargonidin per 100 g sample.
Sample 1
Sample 2
Sample 3
Abs at pH 1.0
Abs at pH 4.5
mg pelargonidin per 100 g sample
Results
See the Results excel file.
References:
1. Fennema, O.R. 1996 Food Chemistry 3rd Ed. Marcel Dekker, Inc. New York.
2. Aurand, L.W. and A.E. Woods 1977. Laboratory Manual in Food Chemistry AVI Publishing Co., Westport, CT.
3. http://eng.ege.edu.tr/~otles/ColorScience/anthocyanins.htm
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